I want to analyze the bacterial cell membrane damage via TEM. For the preparation of sample of TEM which chemicals are used? I have read the literature and there are different chemicals.
After fixation with glutaraldehyde and washing with different series of ethanol in ascending order and drying the pellet.
Is the next step is using osmium or better to use acetonitrile and infilter with quetol epoxy resin?
- A fast way to see damaged membrane in TEM is to do negative staining:
You make your sample to adhere at the surface of a coated EM grid (formvar for example) then you contrast with uranyl acetate before drying.
You can see the morphology of bacteria and you may see blobs at the surface of bacteria indicating pores and leak of cytoplasm.
- SEM can be also useful and is medium time preparation. It is like the negative staining (morphology and blobs) but in addition you see details of the surface of the bacteria (pores).
You make your sample to adhere at the surface of glass coverslip, you contrast with osmium tetroxide (optional), dehydrate with ethanol then you dry with a critical point dryer or with hexamethyldisilasane. Metal sputtering before observation.
- The longest way is resin embedding and cutting for TEM, taking a week:
After fixation, you contrast with osmium tetroxide then uranyl acetate (optional). You dehydrate with ethanol then you embed in resin (epon for example). After polymerization, you ultracut the block and you contrast the sections on EM grids (uranyl acetate and lead citrate).
For dehydration, acetone or acetonitrile can be used instead of ethanol.
Dear Nicolas Barois
I have a problem with the drying of the sample because our laboratory does not have the critical point technique. Please inform me of the temperature and pressure at which drying can be carried out using a vacuum glass oven?
thank you
Hello KhadijaZaki,
Use hexamethyldisilazane (HMD) under a hood.
After the dehydration with 100% ethanol, wash with HMDS, remove the excess of HMDS and let evaporate the sample at room temperature overnight.
Nicolas Barois thank you so much for guiding about the slide preparation for TEM. I want to ask whether osmium tetraoxide is necessary for TEM or SEM visualization? As I read the articles and they did not mention this chemical. For reference I am attaching these articles. Please guide me.
Noor Ul Ain
I see that in your second reference, authors didn't use osmium tetroxide for TEM (resin embedding). They just contrast sections.
Maybe osmium tetroxide is not necessary for bacteria but... hum... morphology is not top quality (E. coli?... are unrecognizable).
I will say that authors made a fast preparation of bacteria for TEM embedding and sectioning. Hum... fast preparation? The quetol epoxy resin doesn't need dehydration of the sample but authors made dehydration.
For SEM, osmium tetroxide is optional. Personally I always use it. It give more contrast and can harden fragile samples (maybe useless for bacteria).
Anyway if you go for osmium tetroxide, it is very toxic, so always use it under the hood with gloves.
You may also consider the use of potassium permanganate, less harmful than osmium tetroxide.
previous discussion:
https://www.researchgate.net/post/The_use_of_potassium_permanganate_KMnO4_as_an_electron-dense_stain_provide_a_adequate_contrast_in_electron_or_x-ray_microscopy?_ec=topicPostOverviewFollowedQuestions&_sg=3HSeFAjDWldH3Z_-pw1aEmL_dBB4pgCcCwageul-cU_wyUFfmgGKD5kUGViUiTZQH7b0DLAN5VJe40Fq
Nicolas Barois thank you so much for helping me out and guiding me for the preparation of sample.
- Badges
- Science topic
- Similar topics
- Chemistry
- Nanotechnology
- Nano